The worldwide demand for high potency sweeteners is increasing and, with blending of different sweeteners becoming a standard practice, the demand for alternatives is expected to increase. The sweet herb of Paraguay, Stevia rebaudiana Bertoni, produces an alternative sweetener with the added advantage that Stevia sweeteners are natural plant products. In addition, the sweet steviol glycosides have functional and sensory properties superior to those of many high potency sweeteners.
Stevia rebaudiana Bert. is one of 154 members of the genus Stevia and one of only two that produce sweet steviol glycosides. There is a large effort aimed at establishing Stevia as a crop in Japan as well as a number of other countries. However, no large scale mechanized production has been established and Stevia sweeteners are not yet found in mainstream food products in most countries of the world.
Stevia is a typical member of the Compositae. It is a small shrubby perennial growing up to 65 cm tall, and appears to be self-incompatible. The results of a complete diallel cross with 8 parents found the amount of selfing to range between 0 and 0.5%, while outcrossing ranged from 0.7 to 68.7%, indicating that some form of self-incompatibility system is operating (Katayama et al. 1976). Stevia is diploid and has 11 chromosomes, which is characteristic for most of the South American members of the genus.
The sweet diterpene glycosides of Stevia have been characterized and eight sweet glycosides of steviol have been identified. These glycosides accumulate in Stevia leaves where they may comprise from 10 to 20% of the leaf dry weight. On a dry weight basis, a typical profile for the four major glycosides found in the leaves of Stevia comprises 0.3% dulcoside, 0.6% rebaudioside C, 3.8% rebaudioside A and 9.1% stevioside. Other glycosides identified within Stevia include rebaudioside B, C, and E, and dulcosides A and B. Rebaudioside B may be an artifact formed from rebaudioside A during extraction since both rebaudioside A and rebaudioside D are found to convert to rebaudioside B by alkaline hydrolysis.
Of the four major diterpene glycoside sweeteners present in Stevia leaves only two, stevioside and rebaudioside A, have had their physical and sensory properties well characterized. Stevioside and rebaudioside A were tested for stability in carbonated beverages and found to be both heat and pH stable (Chang and Cook, 1983). Stevioside is between 110 and 270 times sweeter than sucrose, rebaudioside A between 150 and 320 times sweeter than sucrose, and rebaudioside C between 40 and 60 times sweeter than sucrose. Dulcoside A was 30 times sweeter than sucrose. Rebaudioside A was the least astringent, the least bitter, had the least persistent aftertaste and was judged to have the most favorable sensory attributes of the four major steviol glycosides (Phillips, 1989 and Tanaka, 1997). Dubois and Stephanson (1984) have also confirmed that rebaudioside A is less bitter than stevioside and demonstrated that the bitter notes in stevioside and rebaudioside A are an inherent property of the compounds and not necessarily the result of impurities in whole plant extracts. Bitterness tends to increase with concentration for both stevioside and rebaudioside A. Both stevioside and rebaudioside A are synergistic in mixtures with other high potency sweeteners such as aspartame and are good candidates for inclusion in blends (Schiffman et al. 1995).
Haga et al (JP 51-131900; 1976) disclose methods for the extraction of glycoside compounds from Stevia using a solvent, Ogawa (JP 55-111768; 1980) disclose methods involving the use of a solvent plus a decolorizing agent, Itagaki and Ito (JP 54-041898; 1979) disclose adsorption chromatography, Uneshi et al. (JP 54-030199; 1977) ion exchange, Matsushita and Kitahara (JP 56-121454; 1981) the selective precipitation of individual glycosides, and Tan and Ueki (JP 06-007108; 1994) teach methods based on ultra-filtration. Typical extraction processes involve aqueous or solvent extraction, followed by ion exchange, precipitation or coagulation with filtration, then crystallization and drying (Phillips 1989).
Plant breeding efforts in Stevia have been largely focused on improving leaf yield and rebaudioside A concentration in the leaves. Cultivar descriptions indicate that sufficient genetic variability exists to make significant genetic gains in leaf yield, rebaudioside A content and the ratio of rebaudioside A to stevioside (Brandle and Rosa 1992; Lee et al.1982; Shizhen 1995; Morita T JP 6-96025, 1987). Nakamura and Tamura (1985) report that the levels of dulcoside A and stevioside and rebaudioside A, and C are positively correlated with each other, while stevioside and rebaudioside A, and dulcoside and rebaudioside C are negatively correlated with each other. Total sweet glycoside concentration in some lines from China has been reported to be as high as 20.5%, and in separate cultivars rebaudioside A to stevioside ratios of 9:1 have been disclosed (Shizhen, 1995; Morita, 1987). But there have been no reports of plants comprising economically significant total steviol glycoside levels (i.e. from, or greater than, about 14%) combined with high ratios (i.e. from greater than about 9.1: 1) of rebaudioside A to stevioside. Such a raw material would permit conventional extraction methods, without the need for recrystalization of individual glycosides, to be used to produce a Stevia sweetener with more that 85% rebaudioside A.
Current propagation protocols of Stevia requires clonal propagation of plants comprising desired levels, and ratios, of sweeteners. A cultivar with a rebaudioside A to stevioside ratio of 0.96: 1, compared to 0.36:1 in the starting material, was developed with total glycosides of 22.4%. However, these plants, aside from having very low rebaudioside A to stevioside ratio, were self incompatible and could only be clonally propagated from cuttings (Lee et al. 1982). Other plants have been developed that exhibited rebaudioside A to stevioside ratio to levels as high as 9.1: 1, but total steviol glycosides were 10.1% (Morita T, JP 6-96025, 1987). Again due to self incompatibility the cultivar could not be reproduced using a seed based production system. The costs associated with clonal propagation limit the general applicability for large scale production of Stevia plants, especially in temperate North America where only annual production is an option.
Therefore, there is a need to develop a Stevia cultivar that is enriched in rebaudioside A, with high total steviol glycoside content and that can be produced using a relatively low cost method based on transplants produced from seed.